Endothelial cells respond to hemodynamic mechanical forces such as shear stress and cyclic strain. Disturbances in these forces are associated with endothelial dysfunction and cardiovascular disease. Endothelial cell-cell junctions have been shown to be an important site for transduction of mechanical forces into intracellular signaling events. Several early responses to shear stress occur through a mechanosensory complex, consisting of PECAM-1, VE-cadherin, and VEGFR2, located at the cell-cell junction. This complex is required for the activation of shear-dependent signaling pathways that mediate both cell alignment and activation of many of the pathways that promote atherosclerosis. In this complex, PECAM-1 is hypothesized to be the primary mechanotransducer, where it is directly stimulated by force to initiate signaling. VE-cadherin functions as an adapter that brings VEGFR2 into proximity with PECAM-1, where VEGFR2 is activated by a src family kinase. Activated VEGFR2 then recruits PI3-kinase and mediates activation of downstream pathways. Recent work has revealed additional functions of these proteins in vascular remodeling and function, and shed new light on their involvement in mechanotransduction.

Mechanical forces from blood flow directly control the physiology of the cardiovascular system (1). The major forces of physiological and pathological interest are wall shear stress, acting on the endothelium, and circumferential strain, acting on both endothelial and smooth muscle cells. A major reason for current interest in these forces by both basic scientists and physicians is that sites of atherosclerosis are non-random, strongly correlating with curved or branched sites in the vasculature which, as a result of their geometry, experience low time-averaged shear stress, shear stress reversal, and spatial and temporal gradients in shear stress (2, 3). While numerous risk factors for cardiovascular disease are known, including high LDL cholesterol, diabetes and circulating inflammatory mediators, these are relatively uniform throughout the circulation. The local nature of atherosclerosis has therefore led to the concept that biomechanical factors are crucial. High blood pressure also affects local cellular homeostasis and contributes to the development of atherosclerosis and other pathologies. Recent advances in this field have led to the identification of candidate mechanoreceptors, the specific proteins which sense and respond to mechanical forces, and development of viable hypotheses concerning their involvement in human disease. This article will review the basic features of mechanotransduction in the vascular system, focusing on molecular mechanisms and their potential exploitation in clinical research.

Endothelial cells, exposed to arterial levels of steady or pulsatile unidirectional shear stress in vitro, typically 10 to 40 dynes/cm2, adopt an anti-inflammatory, anti-thrombotic and anti-proliferative phenotype compared to cells in static culture (4-7). These observations have led to the concept that high shear, typical of straight segments of arteries, protects against atherogenic stimuli. By contrast, low flow, oscillatory flow or other flow patterns that involve changes in flow direction and magnitude induce a pro-inflammatory, pro-thrombotic state characterized by high cell turnover (both proliferation and death) relative to cells in static medium. Thus, flow patterns that mimic those found at atherosclerosis-resistant vs. prone regions of arteries inhibit or promote events involved in development of atherosclerosis, respectively.

Endothelial responses to fluid shear stress are often studied by acute application of flow to naive monolayers of cells. This protocol does not mimic any events in normal physiology but is a convenient and effective way to study mechanisms. The approach is analogous to treating starved cells with a bolus of growth factor to study signaling by growth factor receptors, or replating of suspended cells on extracellular matrix to study integrin signaling. Acute responses to arterial shear stress include release of signaling molecules such as nitric oxide and prostacyclin, activation of GTPases, activation of tyrosine and serine/threonine kinases, and phosphorylation of membrane proteins (8, 9). Released signaling molecules, such as prostacyclin and nitric oxide, are physiologically important as they act on smooth muscle cells to mediate vasorelaxation. However, onset of shear also transiently activates a number of inflammatory pathways, including NF-kB, c-jun N-terminal kinase (JNK), p21-activated kinase (PAK) and reactive oxygen species (ROS).

Endothelial cells exposed to long-term arterial levels of unidirectional shear stress remodel their cytoskeleton and downregulate inflammatory pathways. Remodeling involves cell elongation, alignment of actin stress fibers and focal adhesions in the direction of flow, and movement of the microtubule organizing center toward the downstream side of the nucleus (10, 11). Endothelial cells therefore adapt to unidirectional flow, which appears to be important in the acquisition of a quiescent, atherosclerosis-resistant phenotype.

A second important set of flow responses involves activation of atherosclerosis-protective pathways. Flow activates Erk5, which leads to increased expression of the transcription factor Kruppel-like factor (KLF) 2 (12, 13). This transcription factor induces expression of a large repertoire of atherosclerosis-protective genes, including anti-inflammatory, anti-oxidative and anti-thrombotic proteins. Unlike the inflammatory pathways, this set of signals is sustained in high shear, reaching a maximum only after more than 24 hours.

Previous studies have proposed that cell-cell junctions (14), heterotrimeric G-proteins (15), primary cilia (16), caveolae (17), integrins (18), and the glycocalyx (19) mediate transmission or transduction of force from fluid shear stress into signals that regulate the endothelium. At present, the primary mechanotransducers responsible for activation of the anti-atherosclerotic pathways such as Erk5 and KLF2 are unknown. However, work over the past decade has identified a mechanosensory complex at cell-cell junctions, consisting of PECAM-1, VE-cadherin, and VEGFR2, which is required for the activation of a number of shear-dependent signaling pathways that mediate both cell alignment and activation of many of the pro-atherosclerotic pathways (14). In this model, PECAM-1 is the primary mechanotransducer that is stimulated by force to initiate signaling. VE-cadherin functions as an adapter that brings VEGFR2 into proximity of PECAM-1, thereby facilitating VEGFR2 transactivation by a src family kinase. Activated VEGFR2 recruits PI3-kinase, and mediates activation of Akt and eNOS (14, 20). Discussion of these pathways will be the main subject of the rest of this review.

PECAM-1 is an adhesion and signaling molecule expressed on the surface of endothelial cells, platelets, and leukocytes (21). It is a transmembrane protein with an extracellular region that contains six immunoglobulin-like domains, a transmembrane domain, and a short, highly conserved cytoplasmic tail that contains an immunomodulatory ITIM motif (22) (Fig 2B). This motif consists of two tyrosine residues that upon phosphorylation bind the src homology region 2 domain-containing phosphatase 2 (SHP2), which inhibits a number of activating pathways. In endothelial cells, PECAM-1 primarily localizes to intracellular junctions, where it engages in homophilic, trans interactions with PECAM-1 on neighboring cells. It also localizes to the lateral border recycling compartment (23). PECAM-1 has also been found to bind to a number of other counter receptors including integrin αvβ3 but the function of these interactions is less well characterized (24).

PECAM-1 knockout mice have no major developmental or vascular defects (25, 26). However, they have larger diameter collateral arteries compared to wild-type mice (27). A more pronounced decrease in leukocyte emigration was observed in PECAM-1 knockout in FVB/n mice as compared to the standard C57BL/6 strain, suggesting that C57BL/6 mice can compensate for the lack of PECAM-1 (28). Platelets and leukocytes from PECAM-1-/- mice are hyper-activated, most likely due to the loss of the ITIM function (29, 30). Arterioles from PECAM knockout mice exhibit reduced NO-dependent dilation by shear stress (31). In a model of hind limb ischemia, PECAM knockout mice show reduced collateral remodeling and reduced recovery of perfusion, which was attributed to a lack of endothelial response to shear stress (27).

PECAM-1 knockout mice, when crossed into atherosclerotic mouse models, have altered distributions of atherosclerotic lesions. In both the apolipoprotein E (-/-) and the low density lipoprotein receptor (-/-) background, deletion of PECAM decreased lesion size in the lesser curvature of the aortic arch, a principal region of reversing shear stress (32-34). This effect was due to endothelial PECAM-1 since it was also seen in bone marrow chimeras when the donor was PECAM-1-positive. However, PECAM expression was shown to have no effect on lesions in the descending thoracic and abdominal aorta (33). In low density lipoprotein receptor (-/-) mice, PECAM deletion resulted in enlarged lesions in the aortic sinus, branching arteries, and descending thoracic and abdominal aorta (32). Taken together, these studies suggest that PECAM-1 exerts complex effects depending on the model and the site. The data support the idea that endothelial PECAM-1 is an important pro-atherosclerotic mechanosensor for the aortic arch. However, at other sites it can drive both pro- and anti-atherogenic responses. Its immunomodulatory function in platelets and leukocytes is the primary candidate for the anti-atherogenic effects. The data therefore also suggest that the relative importance of these different pathways differ depending on the location within the vasculature, for reasons that are currently unknown.

Human single nucleotide polymorphisms of PECAM are associated with early onset of atherosclerosis and increased risk of cardiovascular disease. The SNPs fall into two distinct haplotypes L98S536R643 and V98N536G643, the latter polymorphism being more often associated with cardiovascular disease (35). It is unclear the relative importance of each polymorphic site; however, the R643G dimorphism is in the cytoplasmic domain near the first ITIM site, suggesting that this dimorphism could alter PECAM signaling. Interestingly, endothelial PECAM tyrosine phosphorylation was increased in the homozygous VNG as compared to the homozygous LSR (36, 37). The PECAM polymorphisms have also been shown to affect leukocyte binding to and transmigration through ECs (37, 38). It remains unclear if the PECAM polymorphisms result in increased cardiovascular disease through altered endothelial cell mechanoresponsiveness or increased monocyte/macrophage/platelet reactivity.

The cytoplasmic binding partners of PECAM-1 are at present incompletely characterized. PECAM-1 has been shown to interact with β-catenin and γ-catenin (also known as plakoglobin) (39, 40). However, this has not been studied extensively. The ITIM tyrosines (Y663 and Y686) can be phosphorylated by src family kinases and then binds the protein tyrosine phosphatase SHP2. Fyn appears to be the major kinase responsible for ITIM phosphorylation in response to mechanical stimulation (41). Both shear stress and cyclic stretch result in increased tyrosine phosphorylation of PECAM (41, 42). PECAM phosphorylation and its interaction with SHP2 has been implicated in activation of Erk MAP kinase (43), transactivation of VEGFR2 (44) and downstream events in response to flow (42), as well as modulation of platelet and leukocyte reactivity (30, 45, 46). Phosporylation of PECAM ITIM tyrosines is also implicated in shear stress activation of eNOS, an important anti-inflammatory pathway (42, 44). However, this result is complex since deletion of PECAM increases basal eNOS activity and NO production as well as decreasing stimulation of NO production by flow (47).

Several model systems have been used by the Fujiwara group to study mechanotransduction by PECAM. Stretching confluent monolayers of endothelial cells or exposure to hypoosmotic medium that induces cell swelling also increased PECAM-1 tyrosine phosphorylation (43). Interestingly, PECAM was still phosphorylated by cyclic stretch in detergent extracted cells, indicating that PECAM phosphorylation is not dependent on an intact plasma membrane or soluble cytoplasmic components (41). Pulling on PECAM directly with magnetic beads coated with PECAM antibodies also induced tyrosine phosphorylation, similarly to shear stress (43). However, unlike the response to flow where PECAM is essential for actin alignment parallel to the direction of shear stress (14), PECAM knockout endothelial cells align normally in cyclic stretch (perpendicular to the direction of stretch), demonstrating that PECAM-1 is not required for these responses (27). These data were interpreted to mean that multiple means of mechanically stimulating endothelial cells leads to increased force across PECAM-1 and similar downstream signals.

Vascular endothelial (VE) cadherin is a critical component of endothelial adherens junctions and a determinant of vascular permeability in vitro and in vivo (48). The extracellular domain of VE-cadherin engages in homophilic binding both within one cell (cis interaction) and with neighboring cells (trans interaction). As in other cadherins, this domain is stabilized by direct binding of calcium ions. VE-cadherin has a transmembrane domain followed by a cytoplasmic tail that directly associates with β-catenin and γ-catenin (plakoglobin). These proteins ultimately link VE-cadherin to the actin cytoskeleton. Classically, it was thought that α-catenin links β-catenin to actin; however, it has recently been suggested that α-catenin cannot simultaneously bind β-catenin and actin, thus, the cytoskeletal linkage must require other components or mechanisms (49). p120-catenin, also binds to the cytoplasmic domain of VE-cadherin and is thought to regulate cadherin function, stability, and availability at the surface (48, 50). VE-cadherin is necessary for proper vascular development, as its loss or expression of a truncated VE-cadherin lacking the beta-catenin binding site are both embryonic lethal, exhibiting severe vascular abnormalities (51).

Application of mechanical force to cadherins stimulates cellular signaling and actin assembly (52, 53), suggesting that cadherins can act as mechanosensors. Adherens junctions have also been shown to strengthen in response to applied mechanical load (54-56). The specific mechanism for this force-dependent strengthening is not fully undestood, although vasodilator-stimulated phosphoprotein (54), α-catenin (57), vinculin (55), myosin (56), and Rac1 (56) have all been shown to participate.

VE-cadherin is implicated in several aspects of the endothelial responses to fluid shear stress. As mentioned above, it is a component of the junctional complex along with PECAM-1 and VE-cadherin. As such, it is necessary for the transactivation of VEGFR2 and association of the adapter Shc with activated VEGFR2 (58). However, homophilic binding of VE-cadherin is not required for its role in response to shear stress (14). These and other data suggest that VE-cadherin functions mainly as an adapter in this system, bringing PECAM-1 and VEGFR2 together to promote activation of the tyrosine kinase following application of force to PECAM-1. This notion is supported by other studies indicating an association and functional relationship between VE-cadherin and VEGFR2 (51).

VE-cadherin also appears to be a downstream target of shear pathways. Endothelial cells exposed in vitro to onset of flow show a transient disruption of cell-cell junctions coincident with increased permeability of tracers across the cell monolayer, followed by recovery at later times (59). Destabilization was mediated by activation of p21-activated kinase, which stimulated myosin-dependent contraction (59) and also directly phosphorylates VE-cadherin to induce its internalization (60). By contrast, cells exposed to reversing (oscillatory) shear stress show sustained activation of PAK and destabilization of junctions (59). This result correlates with the intermittent VE-cadherin staining at the cell-cell junctions and higher permeability in vivo at regions of disturbed shear (61). These results suggest that shear might alter its own mechanotransducers to modulate cellular responses.

Finally, VE-cadherin has also been implicated in responses to mechanical strain. In this system, stretching ECs on an elastic membrane stimulated cell cycle progression which was dependent on homophilic VE-cadherin adhesion (62). This VE-cadherin-dependent effect of stretch was mediated by activation of the small GTPase Rac1. Whether VE-cadherin is the true force bearing element and mechanotransducer in this system was not established; its involvement as a modifier of the mechanotransduction response is also possible, as in the response to flow. A possible role of VE-cadherin in responses of arteries to blood pressure-induced stretch, however, is still very interesting.

VEGFR2, also known as KDR (in humans) or FLK-1 (in mice), is the transmembrane receptor tyrosine kinase (RTK) that mediates most of the angiogenic effects of VEGF. VEGFR2 consists of an extracellular ligand-binding domain with seven immunoglobulin (Ig)-like motifs, a single transmembrane domain and a cytoplasmic domain that contains a juxtamembrane region, a kinase domain split by a kinase insert, and a carboxyl terminus. Similar to other RTKs, ligand binding induces dimerization of VEGFR2, transphosphorylation of the receptors, and the association of adapter/signaling proteins to the phosphorylation sites. Knockout of VEGFR2 leads to embryonic death at mouse E 8.5-9.5 due to defective endothelial cell differentiation (63).

A number of studies have implicated VEGFR2 in responses of endothelial cells to shear. Shear stimulates ligand-independent phosphorylation of VEGFR2 and activation of downstream pathways such as Akt and MAP kinases (20, 64, 65). Chemical inhibitors reduced the activation of these pathways, production of nitric oxide, and the phosphorylation and translocation to EC junctions of Shc in response to shear stress (58). Tyrosine phosphorylation of Y801 and Y1175 of the VEGFR2 cytoplasmic tail is essential for this shear stress response, mediating its association with PI3-kinase (14). A chemical inhibitor of VEGFR2 also blocked a transient increase in actin stiffness in cells exposed to shear (66). These results may be explained by the participation of VEGFR2 in the junctional complex with PECAM-1 and VE-cadherin, though other roles cannot currently be excluded.

Caveolin has been implicated in these VEGFR2 pathways as well. Knockdown of caveolin-1 attenuated AKT and ERK1/2 activation by both shear stress and VEGF stimulation (67). Indeed, caveolin-1 has been proposed to be a transducer of mechanical signals in endothelial cells (68, 69). However, loss of caveolin-1 results in drastic de-regulation of eNOS and production of NO, among other pathways (70). Thus, it is difficult to determine to what extent results from caveolin-1 knockout or knockdown are due to changes in mechanotransduction vs. changes in endothelial signaling and gene expression that interfere with the responses measured.

Although PECAM-1, VE-cadherin, and VEGFR2 form a mechanosensory complex, almost nothing is known about how these proteins associate, or if there are other proteins associated with the mechanosensory complex. It is notable that N-cadherin, which is also expressed in endothelial cells, does not appear to be able to substitute for VE-cadherin. Thus, VE-cadherin appears to have specific functions not shared with other cadherins. These VE-cadherin specific functions are likely to involve (direct or indirect) interactions with PECAM-1 and VEGFR2. Elucidation of these functions and interactions is a major area for future research.

VEGF-A stimulation was shown to signal through the formation of a VE-cadherin/ β-catenin/PI3-kinase/VEGFR2 complex, similar to the mechanosensory complex (51). There may therefore be a parallel between conventional VEGFR2 signaling and the mechanotransduction pathway, since PI 3-kinase is crucial in both systems. A recent report showed that insulin growth factor-I receptor, E-cadherin, and αv integrin formed a complex with α-catenin, which served as a scaffold (71). These data suggest that synergies between cadherins and growth factor receptors may exist in other systems. Interestingly, a truncated form of VE-cadherin lacking the β-catenin binding region blocked intracellular transmission of the VEGF-A stimulus (51). This result is similar to previous observations that cells lacking β-catenin do not respond to flow (14) and that β-catenin is associated with the mechanosensory complex (14, 65), suggesting that β-catenin is also crucial in both systems.

Another area that needs further exploration is the function of the PECAM cytoplasmic tail. Despite the overwhelming evidence that PECAM is mechanoresponsive, it is difficult to postulate a mechanism for the mechanosensitivity of PECAM if there is no connection to some element of the cytoskeleton. The structure, interactions and mechanosensitity of the PECAM-1 cytosplasmic domain is therefore also an important area.

Finally, it will be important to sort out the multiple functions of PECAM-1 in different cell types in the context of mechanotransduction and atherosclerosis. Tissue-specific, conditional knockout of PECAM-1 in endothelial cells, leukocytes and platelets is needed to resolve the complex results from whole animal knockout studies. Specific mutations that disable individual PECAM functions in mechanotransduction and conventional signaling would also be very useful to address these questions.